cepharanthine and curcumin inhibited mitochondrial ......cepharanthine is a natural alkaloid...

RESEARCH ARTICLE Open Access

Cepharanthine and Curcumin inhibitedmitochondrial apoptosis induced by PCV2Yinlan Xu1†, Jiangang Zheng1†, Panpan Sun2, Jianhua Guo3, Xiaozhong Zheng4, Yaogui Sun1, Kuohai Fan2,Wei Yin1, Hongquan Li1* and Na Sun1*

Abstract

Background: Porcine circovirus type 2 (PCV2) is an immunosuppressive pathogen with high prevalence rate in pigfarms. It has caused serious economic losses to the global pig industry. Due to the rapid mutation of PCV2 strain andco-infection of different genotypes, vaccination could not eradicate the infection of PCV2. It is necessary to screen anddevelop effective new compounds and explore their anti-apoptotic mechanism. The 13 natural compounds werepurchased, with a clear plant origin, chemical structure and content and specific biological activities.

Results: The maximum no-cytotoxic concentration (MNTC) and 50% cytotoxic concentration (CC50) of 13 testedcompounds were obtained by the cytopathologic effect (CPE) assay and (3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method in PK-15 cells. The results of qPCR and Western blot showed that,compared with the PCV2 infected group, the expression of Cap in Paeonol (0.4 mg/mL and 0.2 mg/mL), Cepharanthine(0.003mg/mL, 0.0015mg/mL and 0.00075mg/mL) and Curcumin (0.02 mg/mL, 0.001mg/mL and 0.005mg/mL)treated groups were significantly lowered in a dose-dependent manner. The results of Annexin V-FITC/PI, JC-1, Westernblot and ROS analysis showed that the expression of cleaved caspase-3 and Bax were up-regulated Bcl-2 was down-regulated in Cepharanthine or Curcumin treated groups, while ROS and MMP value were decreased at differentdegrees and the apoptosis rate was reduced. In this study, Ribavirin was used as a positive control.

Conclusions: Paeonol, Cepharanthine and Curcumin have significant antiviral effect. And the PCV2-inducedMitochondrial apoptosis was mainly remitted by Cepharanthine and Curcumin.

Keywords: PCV2, Cap, Paeonol, Cepharanthine, Curcumin, Apoptosis

BackgroundPCV2 is a DNA virus, which is an important pathogencausing high infection rate and immunosuppression inpigs [1]. The diseases caused by PCV2 mainly includepostweaning multisystemic wasting syndrome (PMWS),Porcine dermatitis and nephropathy syndrome (PDNS),Porcine proliferative and necrotizing pneumonia (PNP),etc., which have caused serious economic losses to theglobal pig industry for about 57 years [2]. The capsid

protein (Cap) encoded by the ORF2-encoded gene ofPCV2 is a major viral structural protein and an immu-nogen involving viral replication of PCV2 [3]. Studieshave shown that PCV2 could induce apoptosis and de-crease spleen lymphocytes in immune organs of BALB/cmice and pigs [4, 5] and apoptosis can be inducedthrough endoplasmic reticulum (ER) apoptosis, mito-chondrial apoptosis, reactive oxygen species (ROS) regu-lation, etc. [6–9].It has been reported that vaccination could not eradi-

cate the infection of PCV2, due to the rapid mutation ofPCV2 strain and co-infection of different genotypes, etc.[10, 11]. Therefore, it is needed to control infection.Chinese herbal medicine has the characteristics of high

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* Correspondence: [email protected]; [email protected]†Yinlan Xu and Jiangang Zheng contributed equally to this work.1College of Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi030801, ChinaFull list of author information is available at the end of the article

Xu et al. BMC Veterinary Research (2020) 16:345 https://doi.org/10.1186/s12917-020-02568-0

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efficiency, low toxicity and low residue. It can improvethe immunity, possess anti-stress and anti-virus ability.A variety of natural compounds or their active ingredi-ents have been used to enhance host immune, preventand treat viral diseases, such as Coronavirus diseases[12], Hepatitis B (HB) [13], Infectious bronchitis [14],African swine fever (ASF) [15], Influenza [16] and otherviral diseases. However, the research on the anti-PCV2action of natural compounds and its mechanism has alsobecome a hotspot.In our previous studies, dozens of natural com-

pounds were screened for their antiviral activity, suchas Chlorogenic Acid [17], ligustrazine hydrochloride[18], Scutellarin [17, 19], dipotassium glycyrrhizinate[18, 20, 21], Sodium tanshinone IIA sulfonate [21,22], tea seed saponins [23] and Matrine [19, 24, 25].Among them, only Scutellarin or Matrine had anti-PCV2 effect [19, 25].Therefore, in this study, from many natural com-

pounds with clear chemical structure, traditionalmedicine plant source and specific biological activity,13 compounds with antiviral potential were selectedand tested in the model of PCV2 infected PK-15 cellto screen anti-PCV2 compounds in vitro. Further-more, the potential antiviral mechanism of com-pounds was explored and explained from theperspectives of apoptosis, mitochondrial membranepotential (MMP) and ROS. It provides a theoreticalbasis for the development of a safely, efficiently newcompound with anti-PCV2 effect.

ResultsCytotoxicity of 13 compounds on the PK-15 cells detectedby MTTCompared with the normal group of normal cells, theresults showed that (See Additional file 1), the degree ofcytopathological change were also different after thetreatment of different compounds and different concen-trations of the same compound. Due to the low solubil-ity, the maximum solubility concentration (MSC) of 0.8mg/mL Formononetin, 0.8 mg/mL Icariine and 0.15 mg/mL Astragaloside were used to treat the cells, respect-ively and no change in cell morphology was observed.The MNTC is the same as MSC. The 10 compounds ex-cept Formononetin, Icariine and Astragaloside showeddifferent cytopathic changes (see Additional file 1) andMNTC values (Table 2). Dose-response curves of thetested compounds (Fig. 1) and CC50 value (Table 2) weregenerated by GraphPad Prism 5.0 analysis after calculat-ing the CR value. The curves were “S” type, showing adose-dependent relationship. As the concentration ofthe compound increased, more cells with change inmorphology the was observed.

The anti-PCV2 effect of Paeonol, Cepharanthine andCurcuminTo explore the anti-PCV2 effect of compounds, the ex-pression levels of Cap was detected by qPCR and Westernblot. The results of qPCR showed that, compared to cellcontrol group, the PCV2 copy numbers were significantlyincreased (P < 0.05), compared to PCV2 infected group,the PCV2 copy numbers were significantly decreased inthese compound treated with Formononetin at 0.8, Icar-iine at 0.8, Paeonol at 0.4, 0.2 and 0.1mg/mL, Cephar-anthine at 0.003, 0.0015 and 0.00075mg/mL, Curcumin at0.02, 0.01 and 0.005mg/mL, Curcumol at 0.024 and Syrin-gin at 0.625 and 0.3125mg/mL (P < 0.05) (Fig. 2a).Based on the results by qPCR, dose-dependent Paeo-

nol, Cepharanthine and Curcumin were selected forWestern blot. The results showed that the expressionlevel of Cap protein treated with the three compoundsrespectively were significantly lower than PCV2 infectedgroup (P < 0.05), except the 0.1 mg/mL of Paeonol group(Fig. 2b) (For original and unedited blots see Add-itional file 2). Therefore, the Cepharanthine and Curcu-min were selected for the subsequent anti-apoptotic test.

Inhibition of PCV2-induced apoptosis by Cepharanthineand CurcuminTo evaluate the anti-apoptosis effect of Cepharanthineor Curcumin, Samples were analyzed after being treatedwith Annexin V/PI by Flow cytometer. The resultsshowed that Compared to the PCV2-infected group, thecell apoptosis rates were significantly decreased in thegroup treated with Cepharanthine, Curcumin or Ribavi-rin, demonstrating a dose-dependent response exceptthe group of 0.005 mg/mL Paeonol (P < 0.05) (Fig. 3aand b). Our results indicated that Cepharanthine, Curcu-min or Ribavirin could significantly inhibit PCV2-induced apoptosis (P < 0.05).

Inhibition of PCV2-induced apoptosis by Cepharanthineand Curcumin via the mitochondria pathwayTo evaluate the anti-apoptosis mechanism of Cephar-anthine or Curcumin, Samples were analyzed after beingtreated with JC-1 kit by Flow cytometer. The resultsshowed that Compared to the PCV2-infected group, theMMP value were significantly decreased in the grouptreated with Cepharanthine, Curcumin and Ribavirin,demonstrating a dose-dependent response (P < 0.05)(Fig. 4a and b). The results indicated that Cepharanthine,Curcumin or Ribavirin could significantly inhibit PCV2-induced apoptosis via mitochondrial pathway (P < 0.05).

The mechanism of inhibition of PCV2-inducedmitochondrial apoptosis by Cepharanthine and CurcuminTo evaluate the mechanism of inhibition of PCV2-induced mitochondrial apoptosis by Cepharanthine or

Xu et al. BMC Veterinary Research (2020) 16:345 Page 2 of 11

Curcumin, Samples were analyzed by Western blot andFlow cytometer. The results of Western blot showedthat Compared with the PCV2-infected group, the ex-pression of pro-apoptin cleaved caspase-3 and Bax wassignificantly decreased while the expression of anti-apoptotic protein Bcl-2 was significantly increased inthe treatment group of Cepharanthine, Curcumin orRibavirin (Fig. 5a-h) (The original and unedited blotssee Additional file 2). The results of Flow cytometerafter being stained with ROS kit demonstrated that thevalue of ROS in the Curcumin-treated group showedstrongly downward trend in a dose-dependent manner,compared with the PCV2 control group (Fig. 5i and j).However, only the group of treated with 0.003 mg/mLof Cepharanthine could significantly reduce the amountof ROS (P < 0.05). The results demonstrated thatCepharanthine or Curcumin inhibited the PCV2-induced increase in MMP through the caspase familyand Bcl-2 family mechanisms. Curcumin inhibitedPCV2-induced mitochondrial apoptosis by reducingROS, However, ROS reduction is not the main pathwayfor Cepharanthine and Ribavrin to inhibit PCV2-induced mitochondrial apoptosis.

DiscussionPCV2 is the main pathogen causing porcine circovirusassociated diseases (PCVAD). In this study, 13 naturalcompounds with defined chemical structures, usefulclinical effect and from medicinal plants were selected.After the primary screening, three compounds with anti-viral effect, Paeonol, Cepharanthine and Curcumin werefurther studied. The anti-apoptotic mechanism of thesetwo compounds was investigated by evaluating the levelof key apoptins, MMP and ROS in the cellular mito-chondrial pathway. Ribavirin has a broad antiviral activ-ity and was used as a positive control.Cepharanthine is a natural alkaloid extracted from the

roots of the Stephania japonica, which has anti-inflammatory [26], anti-Hepatitis B virus [27], herpessimplex type-1 virus (HSV-1) [28] and etc. Curcumin isan active ingredient extracted from the rhizome of someplants of the Zingiberaceae and Araceae. Medical re-search shows that curcumin has low toxicity, little ad-verse reactions and multiple therapeutic effects, such asanti-inflammatory [29], anti- oxidative [30], anti-H9N2influenza virus [31], dengue virus [32], hepatitis C virus[33] and so on.

Fig. 1 Dose-response curves of all tested compounds in cytotoxicity assay. 8 of gradient dilutions of each compound was prepared by 2-foldserial dilution and incubated with cells for 60 h. Dose-response curve of the tested compounds used in PK-15 shown the more cytopathic effectwere observed with the increased concentration of compounds


Fig. 2 Anti-PCV2 activities of Paeonol, Cepharanthine or Curcumin detected by qPCR and Western blot. The anti-PCV2 effect of compounds wasevaluated by qPCR and Western blot. Cells were incubated with 104.4 TCID50 of PCV2 for 2 h, then tested compounds were added respectivelyand cultured for 48 h. Cells were collected and relative assay perform. a The expression of the Cap gene was detected by qPCR; b The expressionof the Cap protein was detected by Western blot. GraphPad Prism 5.0 was used for the statistical analysis. All data were presented in Mean ± SEM.Different letters to indicate significant differences between groups (p

Zhang et al. demonstrated that PCV2 Cap could in-duce mitochondrial apoptosis of PK-15 and PAM cellsby interfering with Ca2+ homeostasis, down-regulatingMMP and increasing intracellular ROS [7]; Lin et al.found that PCV2 induced mitochondrial apoptosis ofPCV2 infected PK-15 cells and 293 T cells by increasingthe activities of caspase-3 and caspase-9 and acceleratingthe release of cytochrome C (Cyto C) from mitochondriato cytoplasm [34]; After PCV2 infected RAW264.7 cells,stress-related molecules such as ROS production andNO secretion were significantly increased [35]; Previousstudies have shown that PCV2 infection promotes theaccumulation of ROS, which in turn inhibits the replica-tion of PCV2 through the NF-κB pathway [36]. There-fore, we hypothesized that the experimental compoundscould inhibit the mitochondrial apoptosis of PK-15 cellsinduced by PCV2 infection via acting on mitochondrialmembrane potential. Mitochondria have a central role incell apoptosis. The loss of MMP is the marker of mito-chondrial apoptosis, the changes of MMP are caused byBcl-2 family, Caspase family and Miochondrial perme-ability transition pore (MPTP) [37]. Cleaved caspase-3 is

the main executive protein of apoptosis. There are manymembers of the Bcl-2 family, among which Bax is themain pro-apoptotic protein and Bcl-2 is the main anti-apoptotic protein. The opening and closing of MPTP isinfluenced by ROS, Ca2+, ADP, oxidative stress, high PHvalue and other apoptotic factors [37]. In this study, atfirst, the cell apoptosis and change of MMP were de-tected by flow cytometry after Annexin V/PI doublestaining and JC-1 assay. The results indicated thatCepharanthine, Curcumin or Ribavirin could signifi-cantly inhibit PCV2-induced apoptosis via mitochondrialpathway. To further evaluate the mechanism of Cephar-anthine or Curcumin inhibiting mitochondrial apoptosis,the apoptins of Caspase family and Bcl-2 family were an-alyzed by Western blot, the values of ROS were analyzedby flow cytometry after ROS kit staining. The resultsshowed that Compared with cell control group, the ex-pression levels of cleaved caspase-3 and Bax were up-regulated, and the expression of Bcl-2 was down-regulated, the ROS values are increased. This result isconsistent with the previous reports [6, 34, 38]. Com-pared with PCV2-infected group, the expression levels of

Fig. 3 Inhibition of PCV2-induced cell apoptosis by Cepharanthine or Curcumin. a and b After cells were infected with 104.4 TCID50 of PCV2 for 2h, cells were treated with Cepharanthine, Curcumin or Ribavirin for 48 h. The apoptosis rate was evaluated by Annexin V/PI staining followed byflow cytometry. The data on the graph indicated the total number of cells at the late apoptosis of the right upper quadrant and the earlyapoptosis of the right lower quadrant. The results indicated apoptosis rates were significantly decreased in the treated group with Cepharanthine,Curcumin or Ribavirin Compared to the PCV2-infected group except the group of 0.005 mg/mL Curcumin (P < 0.05)


cleaved caspase-3 and Bax were down-regulated, and theexpression of Bcl-2 was up-regulated in the treated withCepharanthine or Curcumin. The value of ROS wasdecreased but ROS did not decrease in treated with the0.0015 and 0.00075 mg/mL of Cepharanthine. Theresults demonstrated that Cepharanthine or Curcumininhibited the PCV2-induced increase in MMP throughthe caspase family and Bcl-2 family mechanisms.Curcumin could inhibit cell mitochondrial apoptosis byregulating the opening of MPTP through reducing thelevel of ROS, but Cepharanthine and Ribavirin did nothave the similar effect. It is speculated that variousantiviral compounds could inhibit the cell apoptosis bygoverning MMP through affecting mitochondrial apop-tosis pathway.

ConclusionTo summarize, our study revealed that Paeonol, Cephar-anthine and Curcumin have significant antiviral effect,and Cepharanthine and Curcumin could inhibit PCV2-induced mitochondrial apoptosis by through change theMMP (Fig. 6). The study provides a base line for furtherstudies on the anti-PCV2 effect and anti-apoptosismechanism of Cepharanthine and Curcumin in

experimental animal models infected with PCV2, andmay be applied in clinical treatment as anti-virus com-pounds eventually.

MethodsCells, viruses, antiboties and nature compoundsPCV-free pig kidney endothelial cell line (PK-15) waspreserved in our laboratory. Cells were cultured andpassed in Dulbecco’s modified eagle’s medium (DMEM,Hyclone, USA) containing 10% fetal calf serum (FBS, BI,Israel) (10% DMEM) and maintenance in DMEM con-taining 2% FBS (2% DMEM).The strain of PCV2-SH (GenBank: AY686763.1) was

gifted by Professor Jiang Ping of Nanjing AgriculturalUniversity. Virus was replicated and harvest in PK-15cells infected with PCV2. The titer of 106.4 TCID50/ mLwas determined by Indirect Immunofluorescence (IFA).Antibodies against Cap were purchased from Biorbyt

LLC. (California, Britain) cleaved caspase-3, Bcl-2 andBax were purchased from Abcam (Cambridge, USA);GAPDH and horseradish peroxidase (HRP)-conjugatedsecondary antibodies were purchased from Wuhan Sany-ing Biology Technology Co., Ltd. (Wu Han, China).

Fig. 4 Inhibition of PCV2-induced cell apoptosis by Cepharanthine or Curcumin via the mitochondria pathway. a and b The changes of MMP in eachgroup after being staining with JC-1 kit were detected by flow cytometry. R2 represents the value of MMP. The results indicated MMP values weresignificantly decreased in the treated group with Cepharanthine, Curcumin or Ribavirin Compared to the PCV2-infected group (P < 0.05)


Fig. 5 The mechanism of inhibition of PCV2-induced mitochondrial apoptosis by Cepharanthine or Curcumin. After cells were infected with 104.4

TCID50 of PCV2 for 2 h, cells were treated with Cepharanthine, Curcumin or Ribavirin for 48 h. a-h The expression of the apoptin were analyzed byWestern blot and grayscale analysis. h, i The results of flow cytometry showed that ROS values were significantly decreased in the treated groupwith Cepharanthine at 0.003 and Curcumin at 0.02, 0.01 and 0.005 mg/mL Compared to the PCV2-infected group (P < 0.05)


Ribavirin, the positive control drug, was purchasedfrom Beijing Solarbio technology co., LTD; Syringin waspurchased from Nanjing Puyi biotechnology co., LTD;Glycyrrhizin aid was purchased from DOSF biotechnol-ogy co., LTD; Other tested compounds, such as Curcu-mol, Paeonol, Oxymatrine, Caffeic acid, Formononetin,Cepharanthine, Apigenin, Psoralen, Lcariine, Curcuminand Astragaloside were purchased from National Insti-tutes for Food and Drug Control. Compounds informa-tion and chemical structure were respectively listedTable 1 and Fig. 7.

Cytotoxicity assayThe viability of PK-15 cell was assessed with MTT.1.0 × 106 cells/mL of cells were seeded into 96-wellplates and incubated until 100% confluency was reached.The tested compounds were added with two-fold serialdiluted and cultured for another 60 h and cell controlgroup was established. Four repeated wells were set upfor each concentration with 100 μL per well. The cyto-pathic condition of cells was observed every day andphotographed. The compounds and dissolution methodsare shown in Table 1. Then 25 μL MTT (4mg/mL inPBS) was added to each well and incubated at 37 °C for4 h. 150 μL of DMSO (Solarbio, Beijing) was added andincubated for 30 min after removing the culturemedium. The optical density (OD) were measured at490 nm using a microplate spectrophotometer (SpectraMax M5, Molecular Devices, USA). Cytopathic ratio(CR) was calculated based on CR = [(OD control - ODtest)/ OD] × 100. Then MNTC and CC50 on PK-15 cellswere calculated using GraphPad Prism™ 5.0 (Inc. Califor-nia, USA).

Real time quantitative PCRThe anti-PCV2 effect of compounds in PK-15 cell wasdetermined by real time quantitative PCR (qPCR). Whencell confluency rate of 24-well plate reached 80%, 104.4

TCID50 of PCV2 was incubated for 2 h and discardedthe supernatant. MNTC of every test compound was intwo-fold serial dilution with 2% DMEM into 3 gradientsand added to 24-well plates (500 μL/well) (Table 2).Meanwhile, the cell control group, PCV2-infected groupand Ribavirin positive control group were applied andincubated for 48 h with 5% CO2 at 37 °C. DNA was ex-tracted according to the instruction of DNA extractionkit (TianGen, Beijing, China) and the DNA concentra-tion was measured using a nucleic acid concentrationanalyzer (NanoDrop Technologies, Wilmington, DE,USA). The copy number of the Cap gene was detectedby qPCR. The Primers 5′ TAC ATT TCC AGC AGTTTG and 5’CTC CCG CCA TAC CAT AA were usedfor the amplification of 148 bp fragment.

Western blotThe anti-PCV2 effect and anti-apoptotic mechanism ofthe compound was detected by Western blot. When cellconfluency of 6-well plate reached 80%, 104.4 TCID50 ofPCV2 was incubated for 2 h and discarded the super-natant. Paeonol, Cepharaanthine and Curcumin wereadded in turn, 2 mL/well. The cell control group, PCV2-infected group and Ribavirin positive control group wereapplied and incubated for 48 h. Total cell protein wasextracted and protein concentration was determinedusing the BCA protein assay kit (Beyotime Biotechnol-ogy, Jiangsu, China). The protein samples were separatedby SDS-PAGE, then transferred to the PVDF membrane.The levels of PCV2 Cap protein and apoptin were

Fig. 6 The PCV2-induced Mitochondrial apoptosis was mainly remitted by Cepharanthine and Curcumin. The “red arrow” represents thepromoting effect and the red “T-shape” indicates the inhibiting effect. The PCV2 replication is inhibited by Cepharanthine and Curcumin, selectedfrom 13 natural compounds, through mitochondrial apoptosis pathway


Table 1 The compounds used in the test

No. Chemical name Classification Main plant source Cosolvent Purity

1 Formononetin Phenolic acids Mango 1%DMSO 98.10%

2 Icariine Flavonoid glycosides Herba epimedii 1%DMSO 94.20%

3 Astragaloside Saponins Astragalus membranaceus 1%DMSO 96.90%

4 Paeonol Monoterpenes Peony 1%DMSO 99.90%

5 Cepharanthine Alkaloide Stephania 1%DMSO 100.00%

6 Curcumin Polyphenols Turmeric 1%DMSO 98.70%

7 Curcumol Hemiketal Curcuma zedoary 1%DMSO 99.90%

8 Syringin Phenylpropanolides Lilac DMEM 95.20%

9 Oxymatrine Alkaloid Sophora DMEM 92.50%

10 Caffeic acid Organic acid Caffeic 1%DMSO 99.70%

11 Apigenin Flavonoid Apium graveolens 1%DMSO 99.20%

12 Glycyrrhizic acid Triterpenes Glycyrrhiza DMEM 95.00%

13 Psoralen Furancoumarins Fructus psoraleae 1%DMSO 99.70%

Fig. 7 The chemical structure of 13 natural compounds


detected using an eECL Western Blot detection kit(Cwbio Inc., Beijing, China) and chemiluminescence im-aging system (BIO-RAD, California, USA).

Annexin V/PI staining for apoptosisThe anti-apoptotic effect of Cepharaanthine and Curcu-min were detected by Annexin V/PI. Cells in 6-well platewere incubated with 104.4 TCID50 of PCV2 for 2 h anddiscarded the supernatant, then Cepharaanthine andCurcumin were added, the cell control group, PCV2-infected group and Ribavirin positive control group wereapplied and incubated for 48 h. Samples were collected,centrifugated and treated using Annexin V/PI kit (Key-gen Biotech, Nanjing, China), then analyzed by flow cy-tometry (BD Biosciences, USA).

Detection of MMP by JC-1Mitochondrial membrane potential (MMP) of cells wasdetected by JC-1. Cells in the 6-well plate were incu-bated with 104.4 TCID50 of PCV2 for 2 h and discardedthe supernatant, then Cepharaanthine and Curcuminwere added. After 48 h incubation, cells were stainedwith JC-1 using a commercial kit (Beyotime Biotechnol-ogy, Jangsu, China) treated and analyzed by flow cytom-etry (BD Biosciences, USA).

Detection of ROS by flow cytometer assayThe amount of reactive oxygen species (ROS) was mea-sured by flow cytometry. Cells in the 6-well plate wereincubated with 104.4 TCID50 of PCV2 for 2 h and dis-carded the supernatant, Cepharaanthine and Curcuminwere added and incubated for 48 h. Cells were treatedwith ROS assay kit and the changes of ROS were de-tected by Flow cytometer.

Statistical analysisCC50 was calculated using nonlinear regression and theresults of “log (inhibitor) vs. response-variable slope”were analyzed using GraphPad Prism. The gray intensityof protein bolts was analyzed by Image J (National Insti-tutes of Health, USA). Data generated were analyzedusing “Bonferroni: Compare all pairs of columns” in theGraphPad Prism™ 5.0 software for one-way ANOVA im-plemented (GraphPad Software, Inc. California, USA),and were expressed as the mean ± standard error of themean (SEM) of at least 3 repeated experiments. Differentletters (a, b, c, d, etc.) indicate statistically significant dif-ference to other groups (p0.8

2 Icariine 0.8 >0.8

3 Astragaloside 0.15 >0.15

4 Paeonol 0.4 1.2696 ± 0.08983

5 Cepharanthine 0.003 0.008048 ± 0.0006136

6 Curcumin 0.02 0.06296 ± 0.02424

7 Curcumol 0.024 0.115 ± 0.02331

8 Syringin 0.625 1.128 ± 0.5339

9 Oxymatrine 2 8.31 ± 0.3896

10 Caffeic acid 0.0125 0.2581 ± 0.07863

11 Apigenin 0.01 0.04817 ± 0.01821

12 Glycyrrhizic acid 0.25 0.7162 ± 0.04979

13 Psoralen 0.012 0.05549 ± 0.01850


https://doi.org/10.1186/s12917-020-02568-0https://doi.org/10.1186/s12917-020-02568-0

Author details1College of Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi030801, China. 2Laboratory Animal Center, Shanxi Agricultural University,Taigu 030801, Shanxi, China. 3Department of Veterinary Pathobiology,Schubot Exotic Bird Health Center, Texas A&M University, College Station,Texas, TX 77843, USA. 4Medical Research Council (MRC) Centre forInflammation Research, Queen’s Medical Research Institute, The University ofEdinburgh, Edinburgh EH16 4TJ, UK.

Received: 28 April 2020 Accepted: 11 September 2020

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AbstractBackgroundResultsConclusions

BackgroundResultsCytotoxicity of 13 compounds on the PK-15 cells detected by MTTThe anti-PCV2 effect of Paeonol, Cepharanthine and CurcuminInhibition of PCV2-induced apoptosis by Cepharanthine and CurcuminInhibition of PCV2-induced apoptosis by Cepharanthine and Curcumin via the mitochondria pathwayThe mechanism of inhibition of PCV2-induced mitochondrial apoptosis by Cepharanthine and Curcumin

DiscussionConclusionMethodsCells, viruses, antiboties and nature compoundsCytotoxicity assayReal time quantitative PCRWestern blotAnnexin V/PI staining for apoptosisDetection of MMP by JC-1Detection of ROS by flow cytometer assayStatistical analysis

Supplementary informationAbbreviationsAcknowledgementsAuthors’ contributionsFundingAvailability of data and materialsEthics approval and consent to participateConsent for publicationCompeting interestsAuthor detailsReferencesPublisher’s Note

cepharanthine and curcumin inhibited mitochondrial ......cepharanthine is a natural alkaloid...

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